A living body is protected from foreign substances mainly by an immune response, and an immune system has been established by various cells and the soluble factors produced thereby. Among them, leukocytes, especially lymphocytes, play a key role. The lymphocytes are classified in two major types, B lymphocyte (which may be hereinafter referred to as B cell) and T lymphocyte (which may be hereinafter referred to as T cell), both of which specifically recognize an antigen and act on the antigen to protect the living body.
T cell is subclassified to helper T cell having CD (Cluster of Differentiation)4 marker (hereinafter referred to as TH), mainly involved in assisting in antibody production and induction of various immune responses, and cytotoxic T cell having CD8 marker (Tc: cytotoxic T lymphocyte, also referred to as killer T cell, which may be hereinafter referred to as CTL), mainly exhibiting a cytotoxic activity. CTL, which plays the most important role in recognizing, destroying and eliminating tumor cell, virus-infected cell or the like, does not produce an antibody specifically reacting with an antigen like B cell, but directly recognizes and acts on antigens (antigenic peptide) from a target cell which is associated with major histocompatibility complex [MHC, which may be also referred to as human leukocyte antigen (HLA) in human] Class I molecules existing on the surface of the target cell membrane. At this time, T cell receptor (hereinafter referred to as TCR) existing on the surface of the CTL membrane specifically recognizes the above-mentioned antigenic peptides and MHC Class I molecules, and determines whether the antigenic peptide is autologous or nonautologous. Target cell which has been determined to be nonautologous is then specifically destroyed and eliminated by CTL.
Recent years, a therapy which would cause a heavy physical burden on a patient, such as pharmacotherapy and radiotherapy, has been reconsidered, and an interest has increased in an immunotherapy with a light physical burden on a patient. Especially, there has been remarked an effectiveness of adoptive immunotherapy in which CTL capable of specifically reacting with an antigen of interest is induced ex vivo from lymphocyte derived from a human having normal immune function, or the lymphocyte is expanded without induction, and then transferred to a patient. For instance, it has been suggested that in an animal model adoptive immunotherapy is an effective therapy for virus infection and tumor (for example, authored by Greenberg, P. D., published in 1992, Advances in Immunology and Reusser P. and three others, Blood, 1991, 78(5), 1373-1380). In this therapy, it is important to maintain or increase the cell number in a state in which the antigen-specific cytotoxic activity of the CTL is maintained or enhanced.
In the adoptive immunotherapy as described above, it is necessary to administer cytotoxic lymphocytes in the number of cells of a given amount or larger in order to obtain a therapeutic effect. In other words, it can be said that it is the greatest problem to obtain the above number of cells ex vivo in a short period of time.
In order to maintain and enhance an antigen-specific cytotoxic activity of CTL, there has been generally employed a method of repeating stimulation with an antigen of interest when a specific response to an antigen for CTL is induced. However, in this method, the number of CTL finally obtained may usually be decreased, so that a sufficient number of cells cannot be obtained.
As a method for preparing T cell which is effective for the treatment of a disease, there has been known, for instance, adoptive immunotherapy using a lymphokine-activated killer cell (LAK cell) (for example, Rosenberg S. A. et al., N. Engl. J. Med. 1987, 316(15), 889-897) and adoptive immunotherapy using a tumor-infiltrating lymphocyte (TIL) induced with interleukin-2 (IL-2) in a high concentration (for example, Rosenberg S. A. et al., N. Engl. J. Med., 1988, 319(25), 1676-1680 and Ho M. and nine others, Blood, 1993, 81(8), 2093-2101).
Next, regarding the preparation of the antigen-specific CTL, there has been reported a method for isolating and expanding a CMV-specific CTL clone using autologous CMV infected fibroblast and IL-2 (for example, Riddell S. A. and four others, J. Immunol., 1991, 146(8), 2795-2804) or using anti-CD3 monoclonal antibody (anti-CD3 mAb) and IL-2 (for example, Greenberg, P. D. and one other, J. Immunol. Methods, 1990, 128(2), 189-201).
Furthermore, WO 96/06929 discloses an REM method (rapid expansion method). This REM method is a method for expanding a primary T cell population containing antigen-specific CTL and TH in a short period of time. In other words, this method is characterized in that a large amount of T cell can be provided by proliferating individual T cell clones, and that the number of antigen-specific CTL is increased using an anti-CD3 antibody, IL-2, and PBMC (peripheral blood mononuclear cell) made deficient in an ability for proliferation by irradiation, and Epstein-Barr virus (hereinafter simply referred to as EBV)-infected cells.
In addition, WO 97/32970 discloses a modified REM method, wherein the method is a method using as a feeder cell a nondividing mammal cell strain expressing a T-cell stimulating component which is distinguishable from PBMC to reduce an amount of PBMC used.
The lymphokine-activated killer cell (LAK cell) is a functional cell population having a cytotoxic activity, which is obtained by adding IL-2 to peripheral blood (peripheral blood leukocyte), umbilical cord blood, tissue fluid or the like containing lymphocytes, and culturing the cells in vitro for several days. During the culture, proliferation of the LAK cell is further accelerated by adding an anti-CD3 antibody thereto and culturing the cell. The LAK cell thus obtained has a cytotoxic activity non-specifically to various cancer cells and other targets. The LAK cell is also used in the adoptive immunotherapy in the same manner as the above-mentioned CTL.
As described above, utilization of IL-2 is essential in the step of obtaining a cytotoxic lymphocyte, for instance, CTL, LAK cell, TIL or the like. The cell is further activated by binding of IL-2 to interleukin-2 receptor (IL-2R) on a cell surface. In addition, IL-2R has been known as an activation marker for a lymphocyte. From these viewpoints, it is important to improve IL-2R expression on the cell surface. In addition, in the induction of CTL, it is important to improve an efficiency for inducing a precursor cell of CTL subjected to stimulation by an antigen as CTL, i.e., to improve a proportion (ratio) of the CD8-positive cell in a group of cells after the induction.
Usually, serum or plasma is also added thereto in a ratio of 5% by volume to 20% by volume, when these lymphocytes are expanded ex vivo. This serum or plasma is a component required when a cell such as a lymphocyte is cultured ex vivo. However, risk of various virus infections and the like cannot be excluded, since serum or plasma is derived from blood of a nonautologous animal (human, bovid or the like). In addition, it is impossible to completely deny the presence of a virus or a pathogenic microorganism undetectable with current detection technique.
In this regard, in recent years, more and more serum or plasma derived from a patient (autologous serum or plasma) is used. However, it may lead to significant risk for the patient to take a large amount of blood from the patient for obtaining serum or plasma in an amount required for culture, since it causes a heavy physical burden on the patient. In order to avoid this risk, a small amount of serum or plasma is used to expand for obtaining lymphocytes required for treatment, which is to be consequently culture with low concentration of serum or plasma. Generally, growth of cells such as lymphocytes is unstable in the culture under low-serum or low-plasma conditions; thereby cells cannot be obtained in an amount required for the treatment. Furthermore, serum-free culture is strongly required for avoiding the physical burden and the risk of infection as mentioned above. However, most cells cannot grow under such culture conditions.
Therefore, a method for expanding a lymphocyte with low-serum or serum-free (low-plasma or plasma-free) is strongly required.
If a method for expanding a lymphocyte under serum-free (plasma-free) conditions is established, difference in serum or plasma among lots can be eliminated, and negative elements resulting from the serum or plasma from a patient (such as immunosuppressive components) can be excluded, whereby the advantage obtained by the establishment of such system is inestimable.
Fibronectin is a gigantic glycoprotein having a molecular weight of 250 thousands, which exists in an animal blood, on the surface of a cultured cell, or in an extracellular matrix of a tissue, and has been known to have various functions. A domain structure thereof is divided into seven portions (hereinafter refer to FIG. 1), wherein three kinds of similar sequences are contained in an amino acid sequence thereof, repetitions of each of these sequences constituting the entire sequence. Three kinds of the similar sequences are referred to as type I, type II and type III. Among them, the type III is constituted by 71 to 96 amino acid residues, wherein a coincidence ratio of these amino acid residues is 17 to 40%. In fibronectin, there are fourteen type III sequences, among which the 8th, 9th or 10th sequence (each being hereinafter referred to as III-8, III-9 or III-10) is contained in a cell binding domain, and the 12th, 13th or 14th sequence (each being hereinafter referred to as III-12, III-13 or III-14) is contained in a heparin binding domain. In addition, a VLA (very late activation antigen)-5 binding region is contained in III-10, and its core sequence is RGDS. In addition, a region referred to as IIICS exists at a C-terminal side of the heparin binding domain. A region referred to as CS-1 consisting of 25 amino acids and having a binding activity to VLA-4 exists in IIICS (for example, authored by Deane F. Momer, published in 1988, FIBRONECTIN, ACADEMIC PRESS INC., P1-8, Kimizuka F. and eight others, J. Biochem., 1991, 110(2), 284-291 and Hanenberg H. and five others, Human Gene Therapy, 1997, 8(18), 2193-2206).